Project
protocol
-
Contents
Workflow
and
sampling
Step |
Procedure |
Equipment |
Data
collected |
1 |
At
5
wks
of
age,
mice
are
weighed;
randomly
selected
mice
are
switched
to
high-fat
high-sucrose
diet
(HFHS)
(controls
remain
on
standard
chow)
for
a
duration
of
16
wks |
Scales |
Body
weight |
2 |
At
21
wks
of
age,
mice
are
moved
to
a
clean
cage
and
fasted
overnight
(16h) |
- |
- |
3 |
Mice
are
weighed
and
anesthetized |
Scales |
Body
weight |
4 |
Body
length
measured |
Ruler |
Body
length,
body
mass
index
(BMI) |
5 |
Blood
glucose
measured |
Glucometer |
Blood
glucose |
6 |
Plasma
isolated
|
Heparinized
micro-hematocrit
capillary
tubes |
- |
7 |
Lipids,
insulin
levels
measured |
- |
Plasma
cholesterol
and
triglycerides;
plasma
insulin;
HOMA-IR |
8 |
Mice
euthanized
and
liver
harvested
and
prepared
for
analysis |
Scales |
Liver
weight,
liver
triglycerides |
Equipment
and
supplies
- Scales
- One
Touch
Ultra
glucometer
(Lifescan,
Inc.,
Milipitas
CA)
- Plasma
separation
tubes
with
gel
barrier
(Statspin,
Westwood
MA)
- Heparinized
micro-hematocrit
capillary
tubes
(Fisher
Scientific,
Pittsburgh
PA)
- ELISA
(Mercodia,
Winston-Salem
NC)
Reagents
and
solutions
- Standard
chow
(Purina
5010,
Purina
LabDiet,
St.
Louis
MO)
- High-fat
high-sucrose
diet
(Research
Diets
D12331,
New
Brunswick
NJ)
- Avertin
(2,2,2,-tribromoethanol
in
tertiary-amyl
alcohol)
Fisher
Scientific,
Pittsburgh
PA
- Colorimetric
reagents
and
standards
(Pointe
Scientific,
Canton
MI)
- GPO-Trinder
reagent
set
(Pointe
Scientific,
Canton
MI)
- 3M
KOH/65%
ethanol
- Dry
ice
- Liquid
nitrogen
Procedure:
Body
size
and
anesthetization
- Mice
(fasted
overnight
16h)
are
weighed
and
anesthetized
via
intraperitoneal
injection
(i.p.)
with
0.8
mg/g
Avertin.
- Nose-to-anus
length
is
measured
and
used
for
body
mass
index
(BMI)
calculation.
Procedure:
Blood
glucose
- Blood
glucose
is
measured
from
the
tail
vein
using
a
One
Touch
glucometer.
Procedure:
Plasma
preparation
- Blood
from
the
orbital
sinus
is
drawn
into
a
separation
tube
with
gel
barrier
using
a
heparinized
micro-hematocrit
capillary
tube.
- Plasma
is
isolated
and
transferred
to
a
new
tube
and
immediately
frozen
on
dry
ice
and
stored
at
-80°C
until
analysis.
Procedure:
Plasma
lipids
and
insulin
- Plasma
total
cholesterol
and
triglycerides
are
measured
with
colorimetric
reagents
and
standards
according
to
manufacturer's
directions.
- Plasma
insulin
is
measured
with
a
mouse
ultrasensitive
insulin
ELISA
according
to
manufacturer's
directions.
- Homeostasis
model
assessment
insulin
resistance
(HOMA-IR)
is
calculated
as
follows:
HOMA-IR
=
[fasting
insulin
x
fasting
glucose]/22.5.
Procedure:
Liver
weight
and
triglycerides
- Livers
are
weighed
and
frozen
in
liquid
nitrogen.
- Tissue
(100-200
mg)
is
saponified
in
an
equal
volume
by
weight
of
3M
KOH/65%
ethanol
to
convert
triglycerides
to
glycerol
and
fatty
acids.
- Glycerol
is
measured
colorimetrically
using
a
GPO-Trinder
reagent
set
to
quantify
the
triglyceride
content.
- Total
triglycerides
are
estimated
by
multiplying
the
liver
triglyceride
content
by
total
liver
weight.
Data
collected
by
investigator
- body
weight
- body
length
- BMI
- blood
glucose
- plasma
total
cholesterol
- plasma
triglycerides
- plasma
insulin
- HOMA-IR
- liver
weight
- liver
triglycerides
- total
liver
triglycerides
|